RESUMO
Salmonella enterica serovar Typhi (S. Typhi) is a human enteropathogen that can survive in macrophages and cause systemic infection. Autophagy and inflammation are two important immune responses of macrophages that contribute to the elimination of pathogens. However, Salmonella has derived many strategies to evade inflammation and autophagy. This study investigated inflammation-related NF-κB signaling pathways and autophagy in S. Typhi-infected macrophages. RNA-seq and quantitative real-time PCR indicated that mRNA levels of NF-κB signaling pathway and autophagy-related genes were dynamically influenced in S. Typhi-infected macrophages. Western blots revealed that S. Typhi activated the NF-κB signaling pathway and induced the expression of inhibitor protein IκBζ. In addition, S. Typhi enhanced autophagy during early stages of infection and may inhibit autophagy during late stages of infection. Thus, we propose that S. Typhi can influence the NF-κB signaling pathway and autophagy in macrophages.
Assuntos
NF-kappa B , Salmonella typhi , Autofagia , Humanos , Inflamação , Macrófagos/microbiologia , NF-kappa B/genética , Salmonella typhi/genéticaRESUMO
Salmonella enterica serovar Typhi (S. Typhi) porins, OmpC and OmpF, are potent inducers of the immune response against S. Typhi in mice and humans. Vaccination with porins induces the protection against 500 LD50 of S. Typhi, life-lasting bactericidal antibodies and effector T cell responses in mice; however, the nature of the memory T cell compartment and its contribution to protection remains unknown. In this work, we firstly observed that vaccination with porins induces in situ (skin) CD4+ and CD8+ T cell responses. Analysis of the porin-specific functional responses of skin CD4+ and CD8+ T cells showed IFN-gamma- and IL-17-producing cells in both T cell populations. The memory phenotype of porin-specific T cells indicated the presence of resident and effector memory phenotypes in the skin, and a central memory phenotype in the skin-draining lymph node. In addition, we demonstrated that vaccination with porins via skin reduces the bacterial burden following challenge. Finally, evaluating the role of the circulating T cell memory population in protection, we showed that circulating memory CD4+ and CD8+ T cells are crucial in porin-mediated protection against S. Typhi. Overall, this study highlights the importance of inducing circulating memory T cell responses in order to achieve the optimal protection provided by porins, showing a mechanism that could be sought in the rational development of vaccines.
RESUMO
This study evaluated the impact of feeding regimes on process performance and inactivation of microorganisms during treatment of aquaculture waste with black soldier fly (BSF) larvae. In three treatments (T1-T3), a blend of reclaimed bread and aquaculture waste was used as substrate for BSF larvae. In T1, the substrate was inoculated with four subtypes of Salmonella spp. and Escherichia coli (both at 1% w/w), and offered only once, at the beginning of the 14-day trial. In T2 and T3, the substrate was supplied on three different days, with contaminated substrate provided only the first event in T2 and in all three events in T3. Provision of a lump sum feeding (T1) proved unfavorable for larval growth and process efficiency, but did not affect the microbial reduction effect. The total reduction in Salmonella spp. was approximately 6 log10 in T1 and T2, and 3.3 log10 in T3, while the total reduction in E. coli was approximately 4 log10 in T1 and T2, and 1.9 log10 in T3. After removing the larvae, the treatment residues were re-inoculated with Salmonella spp. and E. coli. It was found that the inactivation in both organisms continued in all treatments that originally contained BSF larvae (T1-T3), suggesting that antimicrobial substances may have been secreted by BSF larvae or by its associated microbiota.
RESUMO
Ciprofloxacin is the choice treatment for infections caused by Salmonella Typhi, however, reduced susceptibility to ciprofloxacin has been reported for this pathogen. Considering the decreased approbation of new antimicrobials and the crisis of resistance, one strategy to combat this problem is to find new targets that enhances the antimicrobial activity for approved antimicrobials. In search of mutants with increased susceptibility to ciprofloxacin; 3,216 EZ-Tn5 transposon mutants of S. Typhi were screened. S. Typhi zxx::EZ-Tn5 mutants susceptible to ciprofloxacin were confirmed by agar diffusion and MIC assays. The genes carrying EZ-Tn5 transposon insertions were sequenced. Null mutants of interrupted genes, as well as inducible genetic constructs, were produced using site-directed mutagenesis, to corroborate phenotypes. SDS-PAGE and Real-time PCR were used to evaluate the expression of proteins and genes, respectively. Five mutants with increased ciprofloxacin susceptibility were found in the screening. The first confirmed mutant was the glutamine synthetase-coding gene glnA. Analysis of outer membrane proteins revealed increased OmpF, a channel for the influx of ciprofloxacin and nalidixic acid, in the glnA mutant. Expression of ompF increased four times in the glnA null mutant compared to WT strain. To understand the relationship between the expression of glnA and ompF, a strain with the glnA gene under control of the tetracycline-inducible Ptet promoter was created, to modulate glnA expression. Induction of glnA decreased expression of ompF, at the same time that reduced susceptibility to ciprofloxacin. Expression of sRNA MicF, a negative regulator of OmpF was reduced to one-fourth in the glnA mutant, compared to WT strain. In addition, expression of glnL and glnG genes (encoding the two-component system NtrC/B that may positively regulate OmpF) were increased in the glnA mutant. Further studies indicate that deletion of glnG decreases susceptibility to CIP, while deletion of micF gene increases susceptibility CIP. Our findings indicate that glnA inactivation promotes ompF expression, that translates into increased OmpF protein, facilitating the entry of ciprofloxacin, thus increasing susceptibility to ciprofloxacin through 2 possible mechanisms.
RESUMO
Chromosomally-mediated reduced susceptibility to ciprofloxacin narrows the therapeutic options in enteric fever. We made a molecular comparison of clinical isolates of fluoroquinolone-resistant strains of Salmonella enterica serotype Typhi from January 2001 to May 2003; 178 isolates were subjected to antimicrobial susceptibility testing by the Kirby-Bauer method of disk diffusion, and agar dilution was used to determine the minimum inhibitory concentration (MIC) to ciprofloxacin. Nalidixic-acid resistant strains (NARST) were observed in 51 percent of the isolates, of which 98.9 percent had decreased susceptibility (MIC>0.125-1mug/mL) to ciprofloxacin. A single strain (4 mug/mL) was resistant to ciprofloxacin and double mutations were found in the gyrA gene (76 Asp->Asn, 44 leu->Ileu). Among seven NARST strains with reduced susceptibility, a single mutation was found in five strains, one of which had 76 Asp->Asn and two each had mutations at 87 Asp->Asn and 72 Phe->Tyr, respectively); no mutations could be detected in two isolates. Routine antimicrobial surveillance, coupled with molecular analysis of fluoroquinolone resistance, is crucial for revision of enteric fever therapeutics.